Monday, September 27, 2010

TRPS1

"Mutations of the human TRPS1 gene cause a dominantly inherited skeletal dysplasia tricho-rhino-phalangeal syndrome (TRPS). Although the gene was identified several years ago, the molecular and cellular mechanisms underlying TRPS are largely unknown. Trps1 is a GATA-type transcription factor that acts as a transcriptional repressor. Recently, we have demonstrated that disruption of the Trps1 gene in mice results in dramatic elongation of growth plates and delayed replacement of cartilage by bone.These abnormalities are accompanied by increased Indian hedgehog (Ihh) expression and elevated Ihh and BMP signaling. Although BMP and Ihh pathways act in parallel to regulate various aspects of endochondral bone formation, it is unclear how they are integrated and controlled at the transcriptional level. The hypothesis of this proposal is that Trps1 constrains the Ihh-BMP positive feedback loop to assure timely progression of chondrocyte maturation and synchronization of chondrocyte development with perichondrial mineralization. We propose studies aimed at elucidating the Trps1 molecular network and its role in regulation of the cross-talk between differentiating chondrocytes and perichondrium. Specifically, we will focus on understanding the mechanisms of delayed cartilage removal and endochondral ossification in Trps1 mutant mice. To address these questions we propose the following specific aims: 1. To understand the molecular and cellular mechanisms of the growth plate elongation caused by disruption of the Trps1 gene. 2. To determine how Trps1 regulates BMP and hedgehog signaling. The first aim will be accomplished by histological and molecular analyses of abnormalities in the growth plate of the mouse model of TRPS. To achieve the second aim we will employ a combination of electrophoretic mobility shift assay (EMSA), reporter expression assays and analyses of the effect of the Trps1 deficiency and over-expression on BMP and hedgehog signaling in a cellular model of chondrogenesis. Additionally, we will test the Trps1 and Ihh genetic antagonism using Trps1;Ihh double mutant mice. Results of the proposed studies will define molecular mechanisms underlying skeletal dysplasia in tricho-rhino-phalangeal syndrome. Importantly, the results of these studies will directly impact our understanding of the transcriptional control of BMP and hedgehog signaling, that are widely involved in the development of multiple organ systems."

"Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate the molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1DeltaGT mice[involves a frame deletion of the binding element of Trps1]). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Indian hedgehog signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated trans-activation. Importantly, generation of Trps1(DeltaGT/+);Runx2(+/-) double heterozygous mice rescued the opposite growth plate phenotypes of single mutants, demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2."

"Trps1 is highly expressed in regions where Runx2 is downregulated"

"in Trps1ΔGT/ΔGT mice, there is a larger zone of cells co-expressing both Ihh and Col10a1[in addition to expanded growth plates], which indicates an increased number of cells in the transition from prehypertrophic to mature hypertrophic stage." However height remained largely the same.

"TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Trps1 [controls] mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. The control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity. "

Addition of Hdac4 didn't affect the proportion of cells in the G2/M phase in the TRPS1+/+ group in contrast to the knockout group where addition of HDAC4 decreased portion of cells in G2/M phase. Thus meaning that overexpression of TRPS1 may not cause skeletal overgrowth. Addition of Hdac4 did alter some H3K9 acetylation parameters however.